Mammalian cloning is possible since 1986, yet the underlying biology remains obscure. Related to this lack of fundamental understanding is the consistently low frequency with which live-born cloned offspring are obtained, irrespective of the species (cattle, goat, pig, sheep and mouse): it is at best approximately 2 percent. To date the majority of efforts have focused on cloning livestock animals, but these large animals are not convenient experimental models. The mouse is particularly suitable for this purpose, because of the ease of handling, maintenance and breeding, the short gestation period, and the advanced state of mouse genetics. But until recently reproducible techniques for mouse cloning did not exist. The overall aim of our studies is to understand the biological phenomena associated with reprogramming following nuclear transfer in mice. In preliminary studies, a reproducible method has been developed to clone viable and fertile mice by nuclear transfer. It has been applied to cumulus cells, tail-derived cells, embryonic stem cells; to female as well as male mice; and to an inbred strain, 129/Ola. Evidence has been provided that cells at various stages of the cell cycle can be used as nucleus donors. Cell lines with embryonic stem-cell properties have been derived from blastocysts generated by nuclear transfer. In total, we have generated greater than 250 live-born cloned mice with this method. The technology has been re-established at The Rockefeller University following a recent move. To test the hypothesis that the nucleus of a differentiated cell in the adult mouse can be reprogrammed by an oocyte and support full development to term of a cloned mouse, and to define thereby the critical parameters that underly the cloning process in mice, the following Specific Aims are proposed: 1) Can nuclei from mature lymphocytes and postmitotic neurons be reprogrammed? 2) Does the stage of the donor cell cycle influence the ability of nuclei to be reprogrammed? 3) How is telomere length affected by reprogramming after nuclear transfer? 4) Can pluripotent ES cell lines be established from blastocysts generated by nuclear transfer? Taken together, these experiments should resolve critical and outstanding issues in the biology of mammalian cloning. They should also validate the choice of the mouse as a model system. The proposed research is expected to improve the efficiency of this technique, although this is not the primary objective.